Robo-mediated filopodium elongation requires a functional Ena–VASP binding site. (A) DRG neurons were transfected with the indicated constructs and stimulated with 100 ng/ml HGF. White arrowheads mark extremely long filopodia observed after HGF stimulation in growth cones expressing Met-Robo or CC3m. Bars, 10 µm. (B) DRG neurons were transfected with the indicated constructs and stimulated with the indicated ligands at time 0. The maximum length of individual filopodia were measured during a 10-min period starting 5 min after ligand addition. Activation of endogenous Robo by Slit elicited similar filopodium elongation in all conditions. (C) Area measurements were generated by manually outlining growth cones just before ligand addition (Apre) and 20 min after stimulation with the indicated ligands (Apost). Fractional change in area (i) is calculated as −(Apre − Apost)/Apre. Negative values indicate a decrease in growth cone area. (ii) Raw growth cone area values. ***, P < 0.0001; n.s., not significant (mean ± SD, one-way ANOVA, n ≥ 6 biological replicates for all conditions).